Characterization of the Asian Citrus Psyllid-' Candidatus Liberibacter Asiaticus' Pathosystem in Saudi Arabia Reveals Two Predominant CLas Lineages and One Asian Citrus Psyllid Vector Haplotype

Abstract

In Saudi Arabia (SA), the citrus greening disease is caused by 'Candidatus Liberibacter asiaticus' (CLas) transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. The origin and route(s) of the ACP-CLas pathosystem invasion in SA have not been studied. Adult ACP were collected from citrus trees in SA and differentiated by analysis of the mitochondrial cytochrome oxidase I (mtCOI) and nuclear copper transporting protein (atox1) genes. A phylogenetic analysis of the Wolbachia spp. surface protein (wsp) gene was used to identify the ACP-associated Wolbachia spp. A phylogenetic analysis of the atox1 and mtCOI gene sequences revealed one predominant ACP haplotype most closely related to the Indian subcontinent founder populations. The detection and identification of CLas in citrus trees were carried out by polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA gene. The CLas-integrated prophage genomes were sequenced, annotated, and used to differentiate CLas populations. The ML and ASTRAL trees reconstructed with prophages type 1 and 2 genome sequences, separately and concatenated, resolved two major lineages, CLas-1 and -2. The CLas-1 clade, reported here for the first time, consisted of isolates from SA isolates and Pakistan. The CLas-2 sequences formed two groups, CLas-2-1 and -2-2, previously the 'Asiatic' and 'Floridian' strains, respectively. Members of CLas-2-1 originated from Southeast Asia, the USA, and other worldwide locations, while CLas-2-2 was identified only in Florida. This study provides the first snapshot into the status of the ACP-CLas pathosystem in SA. In addition, the results provide new insights into the pathosystem coevolution and global invasion histories of two ACP-CLas lineages with a predicted center of origin in South and Southeast Asia, respectively.

Keywords: citrus greening disease; invasive species; phylogeography; prophage genome sequencing.

Figure 1

Phylogeny based on single nucleotide polymorphisms or character states in relation to and geographical distribution of Asian citrus psyllid (ACP) haplotypes in Asia and the Middle East. The phylogeographic breaks (A) are consistent with geographical isolation (B) between haplotypes. The predicted center of origin for ACP is Southeast Asia, where citrus species have also originated, based on genetic analyses and fossil records [56]. Arrows show the inferred hypothetical ancient route of ACP migration leading to the speciation of the phylogeographic haplotypes analyzed here.

Figure 2

Phylogeny of Asian citrus psyllid populations based on the nuclear gene atox1. The psyllid collections from Saudi Arabia formed two clades based on the atox1-B and atox1-C sequences, with atox1-B containing four variants, atox1-B1–4 (blue). The nearly globally distributed ‘invasive’ ACP was represented by one nuclear haplotype, atox1-A, consisting of populations from China and Pakistan (red).

Figure 3

Phylogeny of the Wolbachia surface protein gene (wsp) associated with Wolbachia spp. endosymbionts of Asian citrus psyllid and representative insect species.

Figure 4

Phylogenetic relationships between ‘Candidatus Liberibacter asiaticus’ (CLas) by a maximum likelihood analysis of CLas-associated prophage type1 (A), -prophage type 2 (B), and types 1 and 2 concatenated full-length genome sequences (C). To reconcile the six SC1/type1 and ten SC2/type 2 tree topologies, a CLas species tree was reconstructed using ASTRAL (D). The single and concatenated CLas-prophage sequences are grouped as two clades, CLas-1 and CLas-2. The CLas-2 isolates formed two sister clades, CLas-2-1, which is widely distributed (‘Asiaticus’), and CLas-2-2, which occurs only in Florida (‘Floridian’). A star placed at major nodes indicates robust phylogenetic support.